Cell-autonomous activation of the PI3-kinase pathway initiates endometrial cancer from adult uterine epithelium

Abstract

Epithelial-specific activation of the PI3-kinase pathway is the most common genetic alteration in type I endometrial cancer. In the majority of these tumors, PTEN expression is lost in the epithelium but maintained in tumor stroma. Currently reported PTEN knockout mouse models initiate type I endometrial cancer concomitant with loss of PTEN in both uterine epithelium and stroma. Consequently, the biologic outcome of selectively activating the PI3-kinase pathway in the endometrial epithelium remains unknown. To address this question, we established a malleable in vivo endometrial regeneration system from dissociated murine uterine epithelium and stroma. Regenerated endometrial glands responded to pharmacologic variations in hormonal milieu similar to the native endometrium. Cell-autonomous activation of the PI3-kinase pathway via biallelic loss of PTEN or activation of AKT in adult uterine epithelia in this model was sufficient to initiate endometrial carcinoma. AKT-initiated tumors were serially transplantable, demonstrating permanent genetic changes in uterine epithelia. Immunohistochemistry confirmed loss of PTEN or activation of AKT in regenerated hyperplastic glands that were surrounded by wild-type stroma. We demonstrate that cell-autonomous activation of the PI3-kinase pathway is sufficient for the initiation of endometrial carcinoma in naive adult uterine epithelia. This in vivo model provides an ideal platform for testing the response of endometrial carcinoma to targeted therapy against this common genetic alteration.

Fig. 1.

Dissociated populations of uterine epithelium and stroma form endometrial-like glands in the in vivo regeneration model. (A) Schematic of the endometrial regeneration system. Dissociated endometrial epithelia harvested from adult female mice were combined with cultured uterine stroma obtained from neonatal mice. The collagen plug containing both cellular fractions was implanted subrenally into a SCID mouse. (B) Regenerated tissue contained endometrial-like glands. These glands arose from adult donor epithelium (DsRed transgenic) demonstrated by the expression of fluorescence in Cy3/RFP channel. (C) The marker expression profile of regenerated grafts (Lower) closely resembled the native murine uterus (Upper). The regenerated endometrial glands expressed CK8 in the endometrial epithelium and SMA in the outer wall of the graft. ERα was predominantly expressed in the epithelium and PR was detected both in the epithelium and the stroma. (Scale bars, 100 μm.)

Fig. 2.

Isolated populations of endometrium are highly enriched for uterine epithelial cells. (A) Cell surface markers that define endometrial epithelium vs. stroma. Trop2 is a marker that is solely expressed in the endometrial epithelium. CD90 marks stromal cells adjacent to the uterine epithelium. (B) Isolated adult endometria used in the regeneration assay were predominantly Trop2⁺/CD90⁻ (blue oval, 91% of total isolated cells). Trop2⁺/CD90⁻ cells were epithelial as they gave rise to epithelial colonies in vitro. Trop2⁻/CD90⁺ cells (green oval) were stromal and formed stromal colonies in the 2D in vitro assay. Error bars represent 1 SD. (C) The epithelial nature of the Trop2⁺/CD90⁻ colonies was confirmed with the expression of CK8. In contrast, Trop2⁻/CD90⁺ colonies were CK8⁻ and Vim⁺ or SMA⁺. (Scale bars, 100 μm.)

Fig. 3.

Regenerated endometrial-like glands were functional and responded to hormonal variations. (A) The response of regenerated tissue to alterations in hormonal milieu was similar to normal uterine endometrium. Regenerated glands became hyperplastic in response to unopposed estradiol (E2), secretory with the administration of progesterone pulse (P4), and underwent breakdown after the withdrawal of P4. (B) The regeneration of endometrial glands depends on stromal and epithelial interactions. Combinations of epithelium and stroma led to the formation of normal secretory glands. Implantation of epithelium alone resulted in the formation of atrophic glands, but glands were absent in the stromal grafts. (C) Similar to the native uterus, LIF was detected in regenerated grafts composed of epithelium and stroma. Importantly, expression of LIF was absent in atrophic glands regenerated from epithelium alone. (D) Prolonged exposure to unopposed estrogen resulted in the formation of progressive endometrial hyperplasia. Simple endometrial hyperplasia developed within 8 wk. At 12 wk, complex hyperplasia was seen, which progressed to atypical complex hyperplasia at 16 wk. (Scale bars, 100 μm.)

Fig. 4.

Consequences of cell autonomous PI3-kinase pathway activation in naive adult endometrial epithelium. (A) Activation of AKT in endometrial epithelium led to the development of hyperplasia and well-differentiated adenocarcinoma. Expression of Myr-AKT with a lentiviral vector resulted in the formation of larger grafts comprised of hyperplastic glands compared with controls. Expression of activated AKT was confirmed by immunostaining. Heterogeneous expression of PR was detected in AKT tumors with many areas exhibiting low levels of epithelial PR. (B) AKT initiated tumors result from cell autonomous genetic changes. Primary AKT tumors were dissociated and retransplanted into a secondary graft with WT stroma. The histology of the secondary regenerant was consistent with endometrial adenocarcinoma. (C) Loss of PTEN resulted in formation of well-differentiated endometrial adenocarcinoma. PTEN was absent only in the epithelial compartment of these tumors, recapitulating the PTEN expression pattern observed in human endometrial adenocarcinoma. (Scale bars, 100 μm.)